Vascular endothelial growth factor (VEGF) plays a critical role during normal angiogenesis and also in the pathological angiogenesis that occurs in a number of diseases, including cancer. Initial attempts to block VEGF by using the humanized monoclonal antibody bevacizumab (Avastin, Genentech/Roche), and two kinase inhibitors sorafenib (Nexavar; Bayer) and sunitinib (Sutent, Pfizer) targeting the VEGF receptor (VEGFR) tyrosine kinases are beginning to show promise in human cancer patients, underscoring the importance of optimizing VEGF blockade. The growth of human tumors and development of metastases depend on the de novo formation of blood vessels to reach and provide nutrients for the hypoxic tumor microenvironment. The formation of new blood vessels is tightly regulated by specific growth factors that target receptor tyrosine kinases (RTKs).
VEGF and the Flk-1/KDR RTK have been implicated as the key endothelial cell-specific factor signaling pathway required for pathological angiogenesis, including tumor neovascularization. Inhibition of the VEGF tyrosine kinase signaling pathway blocks new blood vessel formation in growing tumors, leading to stasis or regression of tumor growth. Advances in understanding the biology of angiogenesis have led to the development of several therapeutic modalities for the inhibition of the VEGF tyrosine kinase signaling pathway. A number of these modalities are under investigation in clinical studies to evaluate their potential to treat various forms of human cancer, but the ability of such studies are limited by the fact that local, real time in vivo measurement of the VEGF level and the trends of the VEGF transduction is not readily available.
In normal development VEGF is a crucial regulator of vascular development during embryogenesis (vasculogenesis) and blood-vessel formation in the adult (angiogenesis). In tumor progression, activation of VEGF pathways promotes tumor vascularization, facilitating tumor growth and metastasis. Abnormal VEGF function is also associated with other diseases including atherosclerosis, psoriasis, age related macular degeneration, diabetic blindness, rheumatoid arthritis, and hyperthyroidism. The members of the VEGF and VEGF-receptor protein families have distinct but overlapping ligand-receptor specificities, cell-type expression, and function. VEGF-receptor activation in turn regulates a network of signaling processes in the body that promote endothelial cell growth, migration, and survival.
Biosensors which continuously monitor their surroundings to provide background statistics and warnings against unhealthy conditions are known to be used in medical technology. The following is a brief overview of the prior art including a discussion of some prior art biosensors. Details of the literature are cited by the references noted in the body of this application. There are numerous examples of, for example, gravimetric biosensors. The basis of detection is the decrease in the resonant frequency of a resonator that occurs as analyte species attach to the resonating element. Analyte specificity is conferred for biological analytes by functionalizing (treating) the exposed surface of the resonator with ligands that recognize and bind to the target analyte species. Examples of suitable binding entities for target biological analytes include antibodies, receptors, lectins, aptamers and oligonucleotides.
In one gravimetric biosensor, the immobilized binding group is located in one or more areas on the surface of the membrane whose locations on the membrane, sizes and area immobilization densities are designed to maximize the observed frequency and/or amplitude shifts on target analyte binding and to maximize the discrimination between all combinations of specific and non-specific binding. This discrimination may take three forms: (a) change in resonant frequency of the membrane, (b) appearance or disappearance of higher order harmonic vibrations, or (c) change in amplitude decay rates. In such a biosensor, a single membrane may be comprised of a plurality of individually addressable elements for actuation and for sensing purposes. This technique permits the specific excitement of selected higher order vibrational modes and enables simultaneous vibration actuation of an alarm circuit or like devices. The principles of acoustic wave, sometimes referred to as gravimetric sensors, are well known and applications have appeared in the literature for more than a decade.
Molecular interactions can be detected electronically through the polarizability of biological macromolecules, optically through the use of fluorescencing tags, radiometrically through the use of radioactive labeled tags, or acoustically. Recently, MEMS based sensors have been incorporated in the biotechnical and biomedical fields. Application of acoustic biosensors, range from cell detection, glucose biosensing, antibody-antigen recognition, and protein adsorption.
Piezoelectric quartz crystal microbalances (QCMs) have been used since the late 1950s to detect gas and liquid phase analytes. Application of QCM technology to biological analytes is more recent. QCMs have been used to track the non-specific adsorption of proteins to unmodified and modified quartz crystal surface electrodes. Immobilization of antibodies to the crystal surface confers analyte specificity.
Since the prior art is extensive we have elected to cite some fundamental innovations of the art of biosensors, as it provides for the principles under which most of the advance applications are based on, an example is sampled by Arwin, et al. U.S. Pat. No. 4,072,576 teaches a method for studying biochemical reactions in which a substance, whose activity or concentration is to be determined, affects a substrate specific for the biochemical reaction which includes providing electrodes coated with the substrate, determining as the control value, the capacitance in a measuring device containing the electrodes, introducing the substance into the measuring device, measuring the change in capacitance, and thereby obtaining a quantitative measure of the activity or concentration of the substance present in the sample and affecting the specific substrate on the electrodes.
Schenck U.S. Pat. No. 4,238,757 describes a field effect transistor including conventional source and drain electrodes employs, in the gate region, a layer of antibody specific to a particular antigen. An electrolyte solution such as 0.155 Normal sodium chloride atop the antibody layer provides a predetermined drain current versus drain voltage characteristic for the device. Replacement of the electrolyte solution with another electrolyte solution containing the antigen alters the charge of the protein surface layer due to the antigen-antibody reaction, thus affecting charge concentration in a semiconductor inversion layer in the transistor. The time rate of change of drain current thus provides a measure of the antigenic protein concentration in the replacement solution.
Rice U.S. Pat. No. 4,314,821 describes a method and kit for determining the total amount of an immunologically-reactive substance in a liquid sample containing interfering material capable of binding to an antigen. The method involves the steps of contacting a liquid sample containing or suspected of containing an antibody with the surface of a piezoelectric oscillator having a layer of antigen specific for the antibody attached thereto; washing and drying the oscillator; measuring the resonance frequency of the oscillator; contacting said surface of the oscillator with a liquid reagent containing an excess amount of a substance specifically reactive with all of the antibody bound to the oscillator. Further, washing and drying the oscillator; and measuring the change in resonance frequency of the oscillator form the first measurement whereby the amount of total antibody bound to the oscillator is distinguished from the interfering material bound in earlier step.
Malmros U.S. Pat. No. 4,444,892 introduces a sensor and semiconductor device for determining the concentration of an analyte in a medium. The device features an element constructed of semi conductive organic polymer associated with a binding substance having specific affinity for the analyte.
Iida, et al. U.S. Pat. No. 4,900,423 teaches of technique where an enzyme sensor comprising enzyme acting specifically on a substrate and a transducer for converting the quantitative change of a substance or heat which is produced or consumed during an enzyme reaction to an electrical signal, wherein the enzyme is glucokinase is disclosed. Iida teaches that a determination of an amount of glucose in a sample is possible as well as an accurate determination of adenosine-5′-triphosphate (ATP). A response time of the sensor is almost constant after a long-term use and a decrease of the detecting ratio is very small.